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Background: Severity of lung hypoplasia, pulmonary hypertension (PH) and cardiac dysfunction are major contributors to mortality in congenital diaphragmatic hernia (CDH). Therefore, early assessment and management is important to improve outcome. NT-proBNP is an established cardiac biomarker with only limited data for early postnatal risk assessment in CDH newborns.Aims: To investigate the correlation of NT-proBNP at birth, 6 h, 12 h, 24 h, and 48 h with PH and cardiac dysfunction and the prognostic information of NT-proBNP for the use of ECMO support or mortality.Subjects: 44 CDH newborns treated at our institution (December 2014-October 2017) were prospectively enrolled.Outcome Measures: Primary clinical endpoint was either need for ECMO or death within the first 48 h (group A). Infants not receiving ECMO support were allocated to group B. Mortality was tested as secondary endpoint.Results: NT-proBNP levels measured at 6 h, 12 h, 24 h and 48 h postpartum correlated significantly with PH severity following NICU admission and at 24 h, and with severity of cardiac dysfunction at birth, 24 h, 48 h and after 7 days of life. There was no difference in NT-proBNP levels between survivors and non-survivors. NT-proBNP levels were significantly higher in group A at 6 h (p = 0.007), 12 h (p = 0.036), and 24 h (p = 0.007), but not at birth (p = 0.785) or 48 h (p = 0.15) compared to group B.Conclusion: NT-proBNP analysis in the first 48 h of life may be useful to assess PH and cardiac dysfunction in CDH newborns and to predict the need for ECMO support. [ABSTRACT FROM AUTHOR]

Background: The association between hypocholesterolaemia and outcome of patients with coronary artery disease (CAD) remains controversial. We undertook this study to investigate whether there is an association between spontaneous or under statin therapy occurring hypocholesterolaemia and mortality in patients with CAD. Materials and Methods: This study included 14 952 patients with CAD undergoing percutaneous coronary intervention (PCI). Hypocholesterolaemia was defined as a total cholesterol (TC) <157 mg/dL (the upper limit of 1st quintile of TC). The study outcome was all‐cause mortality at 30 days and 3 years after PCI. Results: Patients are categorized in four groups according to TC and statin therapy on admission: statin‐naïve patients with hypocholesterolaemia (n = 1102), statin‐naïve patients without hypocholesterolaemia (n = 7490), statin‐treated patients with hypocholesterolaemia (n = 1824) and statin‐treated patients without hypocholesterolaemia (n = 4536). In these groups, 30‐day all‐cause deaths occurred in 3.7%, 1.4%, 1.2% and 0.6% of the patients, respectively; 3‐year deaths occurred in 18.0%, 8.4%, 10.9% and 7.2%, of the patients, respectively. After adjustment, hypocholesterolaemia remained independently associated with 30‐day (adjusted hazard ratio [HR] = 1.50, 95% confidence interval [CI] 1.07 to 2.09; P < 0001) and 3‐year (HR = 1.29 [1.12‐1.47]; P <.001) mortality. Statin therapy on admission was independently associated with 30‐day (HR = 0.61 [0.43‐0.86]; P =.012) and 3‐year (HR = 0.82 [0.72‐0.94]; P =.017) mortality with no statin‐by‐cholesterol interaction with respect to 30‐day (adjusted Pint = 0.669) or 3‐year (adjusted Pint = 0.767) all‐cause mortality suggesting that statins reduce the risk of mortality irrespective of cholesterol level. Conclusions: In patients with CAD, hypocholesterolaemia on admission was independently associated with increased risk of all‐cause mortality at 30 days and 3 years after PCI. [ABSTRACT FROM AUTHOR]

Liquid biopsies are revolutionizing the detection and management of malignant diseases. While repetitive DNA sequences, such as LINE-1 and ALU are established in cell-free DNA (cfDNA) research, their clinical applications remain limited. In this study, we explore human satellite 2 (HSATII), a prevalent repeat DNA sequence in plasma that exhibits increased levels in cancer patients, thereby positioning it as a potential pan-cancer biomarker. We employed targeted sequencing and copy number variation (CNV) analysis using two primer pairs to assess the differential abundance of HSATII sequences in the plasma of breast cancer patients compared to healthy individuals. PCR amplicons of HSATII from 10 patients and 10 control subjects were sequenced, generating 151 bp paired-end reads. By constructing a pooled reference dataset, HSATII copy ratios were estimated in the patients. Our analysis revealed several significant CNVs in HSATII, with certain sequences displaying notable gains and losses across all breast cancer patients, suggesting their potential as biomarkers. However, we observed pronounced fragmentation of cfDNA in cancer, leading to the loss of longer PCR amplicons (>180 bp). While not all observed losses can be attributed to fragmentation artifacts, this phenomenon does introduce complexity in interpreting CNV data. Notably, this research marks the first instance of targeted HSATII sequencing in a liquid biopsy context. Our findings lay the groundwork for developing sequencing-based assays to detect differentially represented HSATII sequences, potentially advancing the field of minimally-invasive cancer screening. [ABSTRACT FROM AUTHOR]

Intense physical exercise is known to increase cardiac biomarkers; however, it is unclear, whether this phenomenon is physiological, or if it indicates myocardial tissue injury. The aim of our study was to investigate the effects of seven consecutive days of excessive endurance exercise on continuous assessment of cardiac biomarkers, function, and tissue injury. During a 7‐day trail‐running competition (Transalpine Run, distance 267.4 km, altitude ascent/descent 15556/14450 m), daily blood samples were obtained for cardiac biomarkers (hs‐TnT, NT‐proBNP, and suppression of tumorigenicity‐2 protein (ST2)) at baseline, after each stage and 24–48 h post‐race. In addition, echocardiography was performed every second day, cardiac magnetic resonance imaging (CMR) before (n = 7) and after (n = 16) the race. Twelve (eight males) out of 17 healthy athletes finished all seven stages (average total finish time: 43 ± 8 h). Only NT‐proBNP increased significantly (3.6‐fold, p = 0.009) during the first stage and continued to increase during the race. Hs‐TnT revealed an incremental trend during the first day (2.7‐fold increase, p = 0.098) and remained within the pathological range throughout the race. ST2 levels did not change during the race. All cardiac biomarkers completely returned to physiological levels post‐race. NT‐proBNP kinetics correlated significantly with mild transient reductions in right ventricular function (assessed by TAPSE, tricuspid annular plane systolic function; r = −0.716; p = 0.014). No significant echocardiographic changes in LV dimensions, LV function, or relevant alterations in CMR were observed post‐race. In summary, this study shows that prolonged, repetitive, high‐volume exercise induced a transient, significant increase in NT‐proBNP associated with right ventricular dysfunction without corresponding left ventricular functional or structural impairment. [ABSTRACT FROM AUTHOR]

Children with congenital heart disease (CHD) undergoing cardiac surgery on cardiopulmonary bypass (CPB) are at risk for systemic inflammation leading to endothelial dysfunction associated with increased morbidity. Bioactive adrenomedullin (bio-ADM) is a peptide regulating vascular tone and endothelial permeability. The aim of this study was to evaluate the dynamics of plasma bio-ADM in this patient cohort and its role in capillary leak. Plasma samples from 73 pediatric CHD patients were collected for bio-ADM measurement at five different timepoints (TP) in the pre-, intra-, and post-operative period. The primary endpoint was a net increase in bio-ADM levels after surgery on CPB. Secondary endpoints included association of bio-ADM levels with clinical signs for endothelial dysfunction. Bio-ADM levels increased after surgery on CPB from pre-operative median of 12 pg/mL (IQR [interquartile range] 12.0–14.8 pg/mL) to a maximum post-operative median of 48.8 pg/mL (IQR 34.5–69.6 pg/mL, p<0.001). Bio-ADM concentrations correlated positively with post-operative volume balance, (r=0.341; p=0.005), increased demand for vasoactive medication (duration: r=0.415; p<0.001; quantity: TP3: r=0.415, p<0.001; TP4: r=0.414, p<0.001), and hydrocortisone treatment for vasoplegia (bio-ADM median [IQR]:129.1 [55.4–139.2] pg/mL vs. 37.9 [25.2–64.6] pg/mL; p=0.034). Patients who required pleural effusion drainage revealed higher bio-ADM levels compared to those who did not (median [IQR]: 66.4 [55.4–90.9] pg/mL vs. 40.2 [28.2–57.0] pg/mL; p<0.001). Bio-ADM is elevated in children after cardiac surgery and higher levels correlate with clinical signs of capillary leakage. The peptide should be considered as biomarker for endothelial dysfunction and as potential therapeutic target in this indication. [ABSTRACT FROM AUTHOR]

Simple Summary: In oncology, the identification of early predictors of response/survival is of particular interest. C-reactive protein (CRP) concentrations have been associated with advanced non-small-cell lung cancer and poor prognosis. We characterized the association between anticancer drug exposure, tumor size as a marker of tumor dynamics, and CRP as a marker of inflammation and derived different predictors. CRP at the beginning of treatment cycle 3 (day 42) was identified as the strongest predictor of both progression-free survival and overall survival, and the inflammatory status, monitored by CRP concentration, emerged as a promising prognostic marker. The high significance of longitudinal CRP concentrations compared to baseline concentrations provided a true reflection of the patient status. This framework could be applied to other treatment modalities such as immunotherapies or targeted therapies, allowing the identification of patients at risk of early progression and/or short survival to spare them unnecessary toxicities and offer alternative treatment decisions. In oncology, longitudinal biomarkers reflecting the patient's status and disease evolution can offer reliable predictions of the patient's response to treatment and prognosis. By leveraging clinical data in patients with advanced non-small-cell lung cancer receiving first-line chemotherapy, we aimed to develop a framework combining anticancer drug exposure, tumor dynamics (RECIST criteria), and C-reactive protein (CRP) concentrations, using nonlinear mixed-effects models, to evaluate and quantify by means of parametric time-to-event models the significance of early longitudinal predictors of progression-free survival (PFS) and overall survival (OS). Tumor dynamics was characterized by a tumor size (TS) model accounting for anticancer drug exposure and development of drug resistance. CRP concentrations over time were characterized by a turnover model. An x-fold change in TS from baseline linearly affected CRP production. CRP concentration at treatment cycle 3 (day 42) and the difference between CRP concentration at treatment cycles 3 and 2 were the strongest predictors of PFS and OS. Measuring longitudinal CRP allows for the monitoring of inflammatory levels and, along with its reduction across treatment cycles, presents a promising prognostic marker. This framework could be applied to other treatment modalities such as immunotherapies or targeted therapies allowing the timely identification of patients at risk of early progression and/or short survival to spare them unnecessary toxicities and provide alternative treatment decisions. [ABSTRACT FROM AUTHOR]

Paclitaxel/platinum chemotherapy, the backbone of standard first‐line treatment of advanced non‐small cell lung cancer (NSCLC), exhibits high interpatient variability in treatment response and high toxicity burden. Baseline blood biomarker concentrations and tumor size (sum of diameters) at week 8 relative to baseline (RS8) are widely investigated prognostic factors. However, joint analysis of data on demographic/clinical characteristics, blood biomarker levels, and chemotherapy exposure‐driven early tumor response for improved prediction of overall survival (OS) is clinically not established. We developed a Weibull time‐to‐event model to predict OS, leveraging data from 365 patients receiving paclitaxel/platinum combination chemotherapy once every three weeks for ≤six cycles. A developed tumor growth inhibition model, combining linear tumor growth and first‐order paclitaxel area under the concentration‐time curve‐induced tumor decay, was used to derive individual RS8. The median model‐derived RS8 in all patients was a 20.0% tumor size reduction (range from −78% to +15%). Whereas baseline carcinoembryonic antigen, cytokeratin fragments, and thyroid stimulating hormone levels were not significantly associated with OS in a subset of 221 patients, and lactate dehydrogenase, interleukin‐6 and neutrophil‐to‐lymphocyte ratio levels were significant only in univariate analyses (p value < 0.05); C‐reactive protein (CRP) in combination with RS8 most significantly affected OS (p value < 0.01). Compared to the median population OS of 11.3 months, OS was 128% longer at the 5th percentile levels of both covariates and 60% shorter at their 95th percentiles levels. The combined paclitaxel exposure‐driven RS8 and baseline blood CRP concentrations enables early individual prognostic predictions for different paclitaxel dosing regimens, forming the basis for treatment decision and optimizing paclitaxel/platinum‐based advanced NSCLC chemotherapy. [ABSTRACT FROM AUTHOR]

Fragments of cell-free DNA (cfDNA) in human body fluids often carry disease-specific alterations and are now widely recognized as ideal biomarkers for the detection and monitoring of genomic disorders, especially cancer, that are normally difficult to examine noninvasively. However, the conversion of promising research findings into tools useful in routine clinical testing of cancer has been a slow-moving process. A major reason is that the diagnostic sensitivity and specificity of cfDNA-based clinical assays are negatively impacted by a combination of suboptimal and inter-institutional differences in preanalytical procedures. The most prominent factors include: (i) a poor understanding of the biological factors that determine the characteristics of the cfDNA population in a biospecimen prior to collection, (ii) inattention to how cfDNA with different structures and physical properties are affected differently by a given preanalytical step, and (iii) the sheer number of possible conditions that can be selected from for each preanalytical step along with a continually expanding menu of commercial products that often show varying degrees of bias and efficiency. The convergence of these variables makes it difficult for research groups and institutions to reach a consensus on optimal preanalytical procedures and a challenging task to establish widely applied standards, which ultimately hamper the development of cfDNA assays that are fit for broad clinical implementation. In this review, we follow a systematic approach to explore the most confounding preanalytical factors that affect the outcome of cfDNA measurements. [ABSTRACT FROM AUTHOR]

The article reports that the concept of reference intervals celebrates a big birthday in 2020. It notes that International founded 50 years ago Federation of Clinical Chemistry (IFCC) an "Expert Panel on Reference Values" that help assess laboratory values in health and illness. It adds that no manufacturer could successfully market a test today, without specifying reference limits, and no laboratory may transmit analytical results, checked without these limits too.

Simple Summary: Ovarian cancer is often diagnosed only in advanced stages, which limits therapeutic options and prognosis. Therefore, better and easily accessible methods for the early diagnosis of ovarian cancer are needed. In particular, blood-based biomarkers seem to be promising candidates for the accurate detection of ovarian cancer. We determined the concentrations of the two proteins HMGB1 and sRAGE in the sera of 231 women with ovarian cancer, benign diseases and without known gynecologic disease. In the analyses of receiver operating characteristics, both HMGB1 and sRAGE could distinguish patients with ovarian cancer from healthy women (area under the curve (AUC) 0.77 and 0.65), benign diseases (AUC 0.72 and 0.61) or all non-malignant cases (AUC 0.74 and 0.63). Moreover, the ratio of HMGB1/sRAGE differentiated even better between malignancies and other cases (AUC 0.78, 0.74 and 0.76, respectively). In conclusion, HMGB1 and sRAGE are potential candidates for the development of assays for early diagnosis of ovarian cancer and warrant inclusion in further validation studies. Background: High mobility group box 1 (HMGB1), soluble receptor of advanced glycation end products (sRAGE) and programmed cell death markers PD-1 and PD-L1 are immunogenic serum biomarkers that may serve as novel diagnostic tools for cancer diagnosis. Methods: We investigated the four markers in sera of 231 women, among them 76 with ovarian cancer, 87 with benign diseases and 68 healthy controls, using enzyme immunoassays. Discrimination between groups was calculated using receiver operating characteristic (ROC) curves and sensitivities at fixed 90% and 95% specificities. Results: HMGB1 levels were significantly elevated and sRAGE levels were decreased in cancer patients as compared to benign and healthy controls. In consequence, the ratio of HMGB1 and sRAGE discriminated best between diagnostic groups. The areas under the curve (AUCs) of the ROC curves for differentiation of cancer vs. healthy were 0.77 for HMGB1, 0.65 for sRAGE and 0.78 for the HMGB1/sRAGE ratio, and slightly lower for the differentiation of cancer vs. benigns with 0.72 for HMGB1, 0.61 for sRAGE and 0.74 for the ratio of both. The highest sensitivities for cancer detection at 90% specificity versus benign diseases were achieved using HMGB1 with 41.3% and the HMGB1/sRAGE ratio with 39.2%, followed by sRAGE with 18.9%. PD-1 showed only minor and PD-L1 no power for discrimination between ovarian cancer and benign diseases. Conclusion: HMGB1 and sRAGE have differential diagnostic potential for ovarian cancer detection and warrant inclusion in further validation studies. [ABSTRACT FROM AUTHOR]

Background: Severe coronavirus disease 2019 (COVID-19) disease courses are characterized by immuno-inflammatory, thrombotic, and parenchymal alterations. Prediction of individual COVID-19 disease courses to guide targeted prevention remains challenging. We hypothesized that a distinct serologic signature precedes surges of IL-6/D-dimers in severely affected COVID-19 patients. Methods: We performed longitudinal plasma profiling, including proteome, metabolome, and routine biochemistry, on seven seropositive, well-phenotyped patients with severe COVID-19 referred to the Intensive Care Unit at the German Heart Center. Patient characteristics were: 65 ± 8 years, 29% female, median CRP 285 ± 127 mg/dL, IL-6 367 ± 231 ng/L, D-dimers 7 ± 10 mg/L, and NT-proBNP 2616 ± 3465 ng/L. Results: Based on time-series analyses of patient sera, a prediction model employing feature selection and dimensionality reduction through least absolute shrinkage and selection operator (LASSO) revealed a number of candidate proteins preceding hyperinflammatory immune response (denoted ΔIL-6) and COVID-19 coagulopathy (denoted ΔD-dimers) by 24–48 h. These candidates are involved in biological pathways such as oxidative stress/inflammation (e.g., IL-1alpha, IL-13, MMP9, C-C motif chemokine 23), coagulation/thrombosis/immunoadhesion (e.g., P- and E-selectin), tissue repair (e.g., hepatocyte growth factor), and growth factor response/regulatory pathways (e.g., tyrosine-protein kinase receptor UFO and low-density lipoprotein receptor (LDLR)). The latter are host- or co-receptors that promote SARS-CoV-2 entry into cells in the absence of ACE2. Conclusions: Our novel prediction model identified biological and regulatory candidate networks preceding hyperinflammation and coagulopathy, with the most promising group being the proteins that explain changes in D-dimers. These biomarkers need validation. If causal, our work may help predict disease courses and guide personalized treatment for COVID-19. [ABSTRACT FROM AUTHOR]

Despite recent advances in the treatment of non-small cell lung cancer (NSCLC), acquired drug resistance to targeted therapy remains a major obstacle. Epithelial-mesenchymal transition (EMT) has been identified as a key resistance mechanism in NSCLC. Here, we investigated the mechanistic role of key EMT-regulating small non-coding microRNAs (miRNAs) in sublines of the NSCLC cell line HCC4006 adapted to afatinib, erlotinib, gefitinib, or osimertinib. The most differentially expressed miRNAs derived from extracellular vesicles were associated with EMT, and their predicted target ZEB1 was significantly overexpressed in all resistant cell lines. Transfection of a miR-205-5p mimic partially reversed EMT by inhibiting ZEB1, restoring CDH1 expression, and inhibiting migration in erlotinib-resistant cells. Gene expression of EMT-markers, transcription factors, and miRNAs were correlated during stepwise osimertinib adaptation of HCC4006 cells. Temporally relieving cells of osimertinib reversed transition trends, suggesting that the implementation of treatment pauses could provide prolonged benefits for patients. Our results provide new insights into the contribution of miRNAs to drug-resistant NSCLC harboring EGFR-activating mutations and highlight their role as potential biomarkers and therapeutic targets. [ABSTRACT FROM AUTHOR]

Tumor marker determinations are valuable tools for the guidance of breast cancer patients during the course of disease. They are assessed on diverse analytical platforms that may be associated with differences according to the methods applied and the clinical performance. To investigate the method dependency and clinical significance of breast cancer protein tumor markers, CEA, CA 15-3, CA 125, CA 19-9 and AFP were measured in a total of 154 biobanked samples from 77 patients with breast cancer, 10 with DCIS, 31 with benign breast diseases and 36 healthy controls using a Millipore multiplex biomarker panel (MP) and an automized version of the routinely used Vista LOCI technology. The markers were compared between methods and investigated for diagnostic performance. CEA, CA 15-3 and AFP showed good correlations between both platforms with correlation coefficients of R = 0.85, 0.85 and 0.92, respectively, in all samples, but similarly also in the various subgroups. CA 125 and CA 19-9 showed only moderate correlations (R = 0.71 and 0.56, respectively). Absolute values were significantly higher for CEA, CA 15-3, CA 125 and AFP in the Vista LOCI as compared with the MP method and vice versa for CA 19-9. The diagnostic performance for discrimination of breast cancer from healthy controls was similar for both methods with AUCs in ROC curves for CEA (MP 0.81, 95% CI 0.72–0.91; LOCI 0.81; 95% CI 0.72–0.91) and CA-15-3 (MP 0.75, 95% CI 0.65–0.86; LOCI 0.67, 95% CI 0.54–0.79). Similar results were obtained for the comparison of breast cancer with benign breast diseases regarding CEA (AUC MP 0.62, 95% CI 0.51–0.73; LOCI 0.64, 95% CI 0.53–0.74) and CA-15-3 (MP 0.70, 95% CI 0.6–0.81; LOCI 0.66, 95% CI 0.54–0.77). Both platforms show moderate to good method comparability for tumor markers with similar clinical performance. However, absolute levels in individual patients should be interpreted with care. [ABSTRACT FROM AUTHOR]

Blood-based biomarkers are valuable diagnostic tools for the management of lung cancer patients. They support not only differential diagnosis and histological subtyping, but are also applied for estimation of prognosis, stratification for specific therapies, monitoring of therapy response, surveillance monitoring and early detection of residual or progressive disease. Early diagnosis of lung cancer in high risk populations (screening) is a promising future indication but poses high medical and economic challenges to marker performance. The five mostly used classical ‘tumor markers’ show characteristic profiles of sensitivity and specificity for non-small cell lung cancer (NSCLC) like cytokeratin 19-fragments (CYFRA 21-1), carcino-embryonic antigen (CEA) and squamous cancer cell antigen (SCCA) as well as for small cell lung cancer (SCLC) like progastrin-releasing peptide (ProGRP) and neuron-specific enolase (NSE). Combined use and pattern recognition approaches enable highly accurate diagnosis, subtyping and therapy monitoring. For the interpretation of serial measurements on an individual level, marker-specific algorithms have to be developed. So-called companion diagnostics identify druggable molecular changes in signaling pathways of tumor tissue that can be addressed by targeted therapies. New highly sensitive technologies enable the convenient and serial molecular characterization on circulating tumor DNA (ctDNA) in the blood, too. This approach is helpful when biopsies are not available and to overcome tumor molecular heterogeneity and plasticity. As only a portion of patients have such druggable molecular changes, future strategies will imply the combined use of classical and new ctDNA-based biomarkers to optimize the management of lung cancer patients during the course of disease. [ABSTRACT FROM PUBLISHER]

Notwithstanding some improvement in the earlier detection of patients with lung cancer, most of them still present with a late-stage disease at the time of diagnosis. Next to the most frequently utilized factors affecting the prognosis of lung cancer patients (stage, performance, and age), the recent application of biomarkers obtained by liquid profiling has gained more acceptance. In our study, we aimed to answer these questions: (i) Is the quantification of free-circulating methylated PTGER4 and SHOX2 plasma DNA a useful method for therapy monitoring, and is this also possible for patients treated with different therapy regimens? (ii) Is this approach possible when blood-drawing tubes, which allow for a delayed processing of blood samples, are utilized? Baseline values for mPTGER4 and mSHOX2 do not allow for clear discrimination between different response groups. In contrast, the combination of the methylation values for both genes shows a clear difference between responders vs. non-responders at the time of re-staging. Furthermore, blood drawing into tubes stabilizing the sample allows researchers more flexibility. [ABSTRACT FROM AUTHOR]

The glycoproteins alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) have long been approved as biomarkers for diagnosing and monitoring tumors. International Reference Preparations (IRPs) have been around since 1975. Nevertheless, manufacturer-dependent differences have been reported, indicating a lack of harmonization. This paper analyzes data from 15 external quality assessment (EQA) surveys conducted worldwide between 2018 and 2022. The aim was to gain insight into the longitudinal development of manufacturer-dependent differences for CEA and AFP. In each survey, participating laboratories received two samples with different tumor marker levels. Inter- and intra-assay variability was analyzed and the mean 80% and 90% of the manufacturer collectives were compared to the evaluation criteria of the German Medical Association (RiliBÄK). The median EQA results for CEA revealed manufacturer-dependent differences between the highest and lowest collective of up to 100%; for AFP, the median differences mostly remained below 40%. The coefficients of variation were predominantly low for both markers. We concluded that the current assays for AFP and CEA detection are better harmonized than previously reported. The assays displayed a good robustness; however, a narrowing of the current assessment limits in EQA schemes could further enhance the quality of laboratory testing. [ABSTRACT FROM AUTHOR]

Purpose: Novel biomarkers to better predict outcome and select the best therapeutic strategy for the individual patient are necessary for pancreatic ductal adenocarcinoma (PDAC). Methods: Using a panel assay, multiple biomarkers (IFN-γ, IL-10, IL-6, IL-8, TNF-α, CEA, CA 19–9, CYFRA 21–1, HE4, PD-1 and PD-L1 levels) were measured in serum samples of 162 patients with resected, locally advanced and metastatic PDAC in this retrospective single-center study. Optimal cut-off values to differentiate prognostic subgroups with significantly different overall survival (OS) were determined by receiver operator characteristics and Youden Index analysis. Marker levels were assessed before the start of chemotherapy and correlated with OS by univariate and multivariate Cox analysis. Results: Median OS for resected patients was 28.2 months, for locally advanced patients 17.9 months and for patients with metastatic disease 8.6 months. CYFRA 21–1 and IL-8 discriminated metastatic from locally advanced patients best (AUC 0.85 and AUC 0.81, respectively). In univariate analyses, multiple markers showed prognostic relevance in the various subgroups. However, multivariate Cox models comprised only CYFRA 21–1 in the resected group (HR 1.37, p = 0.015), IL-10 in locally advanced PDAC (HR 10.01, p = 0.014), as well as CYFRA 21–1 and CA 19–9 in metastatic PDAC (p = 0.008 and p = 0.010) as an independent prognostic marker for overall survival. Conclusion: IL-10 levels may have independent prognostic value in locally advanced PDAC, whereas CYFRA 21–1 levels are prognostic after PDAC surgery. CYFRA 21–1 and IL-8 have been identified to best discriminate metastatic from locally advanced patients. [ABSTRACT FROM AUTHOR]

Excellent pre-analytical stability is an essential precondition for reliable molecular profiling of circulating tumor DNA (ctDNA) in oncological diagnostics. Therefore, in vitro degradation of ctDNA and the additional release of contaminating genomic DNA from lysed blood cells must be prevented. Streck Cell-Free DNA blood collection tubes (cfDNA BCTs) have proposed advantages over standard K2EDTA tubes, but mainly have been tested in healthy individuals. Blood was collected from cancer patients (n = 53) suffering from colorectal (n = 21), pancreatic (n = 11), and non-small-cell lung cancer (n = 21) using cfDNA BCT tubes and K2EDTA tubes that were processed immediately or after 3 days (BCTs) or 6 hours (K2EDTA) at room temperature. The cfDNA isolated from these samples was characterized in terms of yield using LINE-1 qPCR; the level of gDNA contamination; and the mutation status of KRAS, NRAS, and EGFR genes using BEAMing ddPCR. CfDNA yield and gDNA levels were comparable in both tube types and were not affected by prolonged storage of blood samples for at least 3 days in cfDNA BCTs or 6 hours in K2EDTA tubes. In addition, biospecimens collected in K2EDTA tubes and cfDNA BCTs stored for up to 3 days demonstrated highly comparable levels of mutational load across all respective cancer patient cohorts and a wide range of concentrations. Our data support the applicability of clinical oncology specimens collected and stored in cfDNA BCTs for up to 3 days for reliable cfDNA and mutation analyses. [ABSTRACT FROM AUTHOR]

Background: Trauma is still a major cause of mortality in people < 50 years of age. Biomarkers are needed to estimate the severity of the condition and the patient outcome. Methods: Cell-free DNA (cfDNA) and further laboratory markers were determined in plasma and serum of 164 patients at time of admission to the emergency room. Among them were 64 patients with severe trauma (Injury Severity Score (ISS) ≥ 16), 51 patients with moderate trauma (ISS < 16) and 49 patients with single fractures (24 femur neck and 25 ankle fractures). Disease severity was objectified by ISS and Glasgow Coma Scale (GCS). Results: cfDNA levels in plasma and serum were significantly higher in patients with severe multiple trauma (SMT) than in those with moderate trauma (p = 0.002, p = 0.003, respectively) or with single fractures (each p < 0.001). CfDNA in plasma and serum correlated very strongly with each other (R = 0.91; p < 0.001). The AUC in ROC curves for identification of SMT patients was 0.76 and 0.74 for cfDNA in plasma and serum, respectively—this was further increased to 0.84 by the combination of cfDNA and hemoglobin. Within the group of multiple trauma patients, cfDNA levels were significantly higher in more severely injured patients and patients with severe traumatic brain injury (GCS ≤ 8 versus GCS > 8). Thirteen (20.3%) of the multiple trauma patients died during the first week after trauma. Levels of cfDNA were significantly higher in non-surviving patients than in survivors (p < 0.001), reaching an AUC of 0.81 for cfDNA in both, plasma and serum, which was further increased by the combination with hemoglobin and leukocytes. Conclusions: cfDNA is valuable for estimation of trauma severity and prognosis of trauma patients. [ABSTRACT FROM AUTHOR]

Background: COVID-19 might pose a risk for adults with congenital heart disease (ACHD). However, data regarding the rate of infection as well as myocardial involvement in ACHD patients are currently lacking. Methods: During the study period from January to June 2021, all consecutive outpatients from our ACHD clinic were eligible to participate. Clinical data were collected. An antibody test for COVID-19 was performed in all patients. Cardiovascular magnetic resonance imaging (CMR) was offered to those with a positive antibody test. Results: Overall, 420 patients (44.8% female, mean age 36.4 ± 11.6 years) participated. Congenital heart defect (CHD) complexity was simple in 96 (22.9%), moderate in 186 (44.3%), complex in 117 (27.9%), and miscellaneous in 21 (5.0%) patients. Altogether, 28 (6.7%) patients had a positive antibody test. Out of these, 14 had an asymptomatic course. The others had mainly mild symptoms and were managed as outpatients. Furthermore, 11 patients (39.3%) had even not been aware of their infection. Fourteen patients underwent a CMR without signs of myocardial involvement in any of them. Conclusions: We observed a number of undetected cases of COVID-19 infections in our ACHD population. Reassuringly, in all cases, the infection had a mild clinical course. [ABSTRACT FROM AUTHOR]

Plasma cell-free DNA (cfDNA) originates from various tissues and cell types and can enable minimally invasive diagnosis, treatment and monitoring of cancer and other diseases. Proper extraction of cfDNA is critical to obtain optimal yields and purity. The goal of this study was to compare the performance of six commercial cfDNA kits to extract pure, high-quality cfDNA from human plasma samples and evaluate the quantity and size profiles of cfDNA extracts—among them, two spin-column based, three magnetic bead-based and two automatic magnetic bead-based methods. Significant differences were observed in the yield of DNA among the different extraction kits (up to 4.3 times), as measured by the Qubit Fluorometer and Bioanalyzer. All kits isolated mostly small fragments corresponding to mono-nucleosomal sizes. The highest yield and reproducibility were obtained by the manual QIAamp Circulating Nucleic Acid Kit and automated MagNA Pure Total NA Isolation Kit. The results highlight the importance of standardizing preanalytical conditions depending on the requirements of the downstream applications. [ABSTRACT FROM AUTHOR]

Body fluids are constantly replenished with a population of genetically diverse cell-free DNA (cfDNA) fragments, representing a vast reservoir of information reflecting real-time changes in the host and metagenome. As many body fluids can be collected non-invasively in a one-off and serial fashion, this reservoir can be tapped to develop assays for the diagnosis, prognosis, and monitoring of wide-ranging pathologies, such as solid tumors, fetal genetic abnormalities, rejected organ transplants, infections, and potentially many others. The translation of cfDNA research into useful clinical tests is gaining momentum, with recent progress being driven by rapidly evolving preanalytical and analytical procedures, integrated bioinformatics, and machine learning algorithms. Yet, despite these spectacular advances, cfDNA remains a very challenging analyte due to its immense heterogeneity and fluctuation in vivo. It is increasingly recognized that high-fidelity reconstruction of the information stored in cfDNA, and in turn the development of tests that are fit for clinical roll-out, requires a much deeper understanding of both the physico-chemical features of cfDNA and the biological, physiological, lifestyle, and environmental factors that modulate it. This is a daunting task, but with significant upsides. In this review we showed how expanded knowledge on cfDNA biology and faithful reverse-engineering of cfDNA samples promises to (i) augment the sensitivity and specificity of existing cfDNA assays; (ii) expand the repertoire of disease-specific cfDNA markers, thereby leading to the development of increasingly powerful assays; (iii) reshape personal molecular medicine; and (iv) have an unprecedented impact on genetics research. [ABSTRACT FROM AUTHOR]

Given the crucial role of mitochondria as the main cellular energy provider and its contribution towards tumor growth, chemoresistance, and cancer cell plasticity, mitochondrial DNA (mtDNA) could serve as a relevant biomarker. Thus, the profiling of mtDNA mutations and copy number variations is receiving increasing attention for its possible role in the early diagnosis and monitoring therapies of human cancers. This applies particularly to highly aggressive pancreatic cancer, which is often diagnosed late and is associated with poor prognosis. As current diagnostic procedures are based on imaging, tissue histology, and protein biomarkers with rather low specificity, tumor-derived mtDNA mutations detected from whole blood represents a potential significant leap forward towards early cancer diagnosis. However, for future routine use in clinical settings it is essential that preanalytics related to the characterization of mtDNA in whole blood are thoroughly standardized, controlled, and subject to proper quality assurance, yet this is largely lacking. Therefore, in this study we carried out a comprehensive preanalytical workup comparing different mtDNA extraction methods and testing important preanalytical steps, such as the use of different blood collection tubes, different storage temperatures, length of storage time, and yields in plasma vs. whole blood. To identify analytical and preanalytical differences, all variables were tested in both healthy subjects and pancreatic carcinoma patients. Our results demonstrated a significant difference between cancer patients and healthy subjects for some preanalytical workflows, while other workflows failed to yield statistically significant differences. This underscores the importance of controlling and standardizing preanalytical procedures in the development of clinical assays based on the measurement of mtDNA. [ABSTRACT FROM AUTHOR]

Unique bits of genetic, biological and pathological information occur in differently sized cell-free DNA (cfDNA) populations. This is a significant discovery, but much of the phenomenon remains to be explored. We investigated cfDNA fragmentation patterns in cultured human bone cancer (143B) cells using increasingly sensitive electrophoresis assays, including four automated microfluidic capillary electrophoresis assays from Agilent, i.e., DNA 1000, High Sensitivity DNA, dsDNA 915 and dsDNA 930, and an optimized manual agarose gel electrophoresis protocol. This comparison showed that (i) as the sensitivity and resolution of the sizing methods increase incrementally, additional nucleosomal multiples are revealed (hepta-nucleosomes were detectable with manual agarose gel electrophoresis), while the estimated size range of high molecular weight (HMW) cfDNA fragments narrow correspondingly; (ii) the cfDNA laddering pattern extends well beyond the 1–3 nucleosomal multiples detected by commonly used methods; and (iii) the modal size of HMW cfDNA populations is exaggerated due to the limited resolving power of electrophoresis, and instead consists of several poly-nucleosomal subpopulations that continue the series of DNA laddering. Furthermore, the most sensitive automated assay used in this study (Agilent dsDNA 930) revealed an exponential decay in the relative contribution of increasingly longer cfDNA populations. This power-law distribution suggests the involvement of a stochastic inter-nucleosomal DNA cleavage process, wherein shorter populations accumulate rapidly as they are fed by the degradation of all larger populations. This may explain why similar size profiles have historically been reported for cfDNA populations originating from different processes, such as apoptosis, necrosis, accidental cell lysis and purported active release. These results not only demonstrate the diversity of size profiles generated by different methods, but also highlight the importance of caution when drawing conclusions on the mechanisms that generate different cfDNA size populations, especially when only a single method is used for sizing. [ABSTRACT FROM AUTHOR]

All cell and tissue types constantly release DNA fragments into human body fluids by various mechanisms including programmed cell death, accidental cell degradation and active extrusion. Particularly, cell-free DNA (cfDNA) in plasma or serum has been utilized for minimally invasive molecular diagnostics. Disease onset or pathological conditions that lead to increased cell death alter the contribution of different tissues to the total pool of cfDNA. Because cfDNA molecules retain cell-type specific epigenetic features, it is possible to infer tissue-of-origin from epigenetic characteristics. Recent research efforts demonstrated that analysis of, e.g., methylation patterns, nucleosome occupancy, and fragmentomics determined the cell- or tissue-of-origin of individual cfDNA molecules. This novel tissue-of origin-analysis enables to estimate the contributions of different tissues to the total cfDNA pool in body fluids and find tissues with increased cell death (pathologic condition), expanding the portfolio of liquid biopsies towards a wide range of pathologies and early diagnosis. In this review, we summarize the currently available tissue-of-origin approaches and point out the next steps towards clinical implementation. [ABSTRACT FROM AUTHOR]

Children with univentricular hearts (UVH) undergo up to three palliative surgical procedures to achieve complete circulatory separation (Fontan circulation). As a marker of cardiac wall stress, NT-proBNP is a promising tool to assess systemic ventricular load in these patients. However, different reference intervals (RI) apply to each stage, as NT-proBNP is highly age-dependent. Children undergoing systemic-to-pulmonary (SP) shunt placement (stage 1), bidirectional cavopulmonary shunt (BCPS, stage 2) or total cavopulmonary connection (TCPC, stage 3) between 2011 and 2021 with NT-proBNP measurement within 7 days before surgery were included. Furthermore, outpatients after TCPC with NT-proBNP measurement were enrolled. Biomarker levels were evaluated using its age-adjusted z-score ("zlog-NT-proBNP"; age-independent RI, −1.96 to +1.96), allowing comparison between different stages and revealing changes in systemic ventricular load independent of the marked physiological decline in RI with age. Overall, 289 children (227 before, 62 after TCPC) met the eligibility criteria. Median time between blood sampling and surgery (SP shunt/BCPS/TCPC) was 2 [1–3] days and 3.2 [2.0–4.5] years after TCPC. Age-adjusted zlog-NT-proBNP levels were 3.47 [2.79–3.93] in children with native UVH (before SP shunt), 3.10 [1.89–3.58] at stage 1 (before BCPS), 1.08 [0.51–1.88] at stage 2 (before TCPC), and 1.09 [0.72–1.75] at stage 3 (after TCPC/Fontan completion). Consequently, BCPS revealed the strongest decrease (median − 2.02 logarithmized standard deviations, p < 0.001). In children with UVH undergoing staged Fontan palliation, zlog-NT-proBNP is a highly promising tool for course assessment of systemic ventricular load, independent of the age-related decline in physiological NT-proBNP concentration. • Age-adjusted zlog-NT-proBNP helps to quantify ventricular stress in children with UVH. • Strongest decrease of zlog-NT-proBNP was observed with bidirectional cavopulmonary shunt. • Children after TCPC mostly reveal physiological zlog-NT-proBNP levels. [ABSTRACT FROM AUTHOR]

* Context.--There is a need to avoid the overdiagnosis of prostate cancer (PCa) and to find more specific biomarkers. Objective.--To evaluate the clinical utility of [-2]pro-prostate-specific antigen ([-2]proPSA) derivatives in detecting clinically significant PCa (csPCa) and to compare it with prostate-specific antigen (PSA) and with the percentage of free PSA (%fPSA). Design.--Two hundred thirty-seven men (PSA: 2-10 ng/mL) scheduled for a prostate biopsy were enrolled. Parametric and nonparametric tests, receiver operating characteristic curves, and logistic regression analysis were applied. Outcomes were csPCa and overall PCa. Results.--Both [-2]proPSA derivatives were significantly higher in csPCa and overall PCa (P < .001). The areas under the curves for the prediction of csPCa were higher for the percentage of [-2]proPSA (%[-2]proPSA) (0.781) and the prostate health index (PHI) (0.814) than for PSA (0.651) and %fPSA (0.724). There was a gain of 11% in diagnostic accuracy when %[-2]proPSA or PHI were added to a base model with PSA and %fPSA. Twenty-five percent to 29% of biopsies could have been spared with %[-2]proPSA (cutoff: ≥1.25%) and PHI (cutoff: ≥27), missing 10% of csPCas. The same results could have been achieved by using [-2]proPSA as a reflex test, when %fPSA was 25% or less (cutoffs: ≥1.12% and ≥24 for %[-2]proPSA and PHI, respectively). Conclusions.--The [-2]proPSA derivatives improve the diagnostic accuracy of csPCa when the PSA value is between 2 and 10 ng/mL, sparing unnecessary biopsies and selecting patients for active surveillance. [-2]proPSA can be used as a reflex test when %fPSA is 25% or less, without reducing the diagnostic accuracy for csPCa and the number of spared biopsies. [ABSTRACT FROM AUTHOR]

Background: Despite the significance of colonoscopy for early diagnosis of colorectal adenocarcinoma (CRC), population-wide screening remains challenging, mainly because of low acceptance rates. Herein, exosomal (exo-miR) and free circulating microRNA (c-miR) may be used as liquid biopsies in CRC to identify individuals at risk. Direct comparison of both compartments has shown inconclusive results, which is why we directly compared a panel of 10 microRNAs in this entity. Methods: Exo-miR and c-miR levels were measured using real-time quantitative PCR after isolation from serum specimens in a cohort of 69 patients. Furthermore, results were compared to established tumor markers CEA and CA 19-9. Results: Direct comparison of exo- and c-miR biopsy results showed significantly higher microRNA levels in the exosomal compartment (p < 0.001). Exo-Let7, exo-miR-16 and exo-miR-23 significantly differed between CRC and healthy controls (all p < 0.05), while no c-miR showed this potential. Sensitivity and specificity can be further enhanced using combinations of multiple exosomal miRNAs. Conclusions: Exosomal microRNA should be considered as a promising biomarker in CRC for future studies. Nonetheless, results may show interference with common comorbidities, which must be taken into account in future studies. [ABSTRACT FROM AUTHOR]

Background Heterozygous familial hypercholesterolemia (FH) represents the most frequent monogenic disorder with an estimated prevalence of 1:250 in the general population. Diagnosis during childhood enables early initiation of preventive measures, reducing the risk of severe consecutive atherosclerotic manifestations. Nevertheless, population-based screening programs for FH are scarce. Methods In the VRONI study, children aged 5–14 years in Bavaria are invited to participate in an FH screening program during regular pediatric visits. The screening is based on low-density lipoprotein cholesterol measurements from capillary blood. If exceeding 130 mg/dl (3.34 mmol/l), i.e. the expected 95th percentile in this age group, subsequent molecular genetic analysis for FH is performed. Children with FH pathogenic variants enter a registry and are treated by specialized pediatricians. Furthermore, qualified training centers offer FH-focused training courses to affected families. For first-degree relatives, reverse cascade screening is recommended to identify and treat affected family members. Results Implementation of VRONI required intensive prearrangements for addressing ethical, educational, data safety, legal and organizational aspects, which will be outlined in this article. Recruitment started in early 2021, within the first months, more than 380 pediatricians screened over 5200 children. Approximately 50 000 children are expected to be enrolled in the VRONI study until 2024. Conclusions VRONI aims to test the feasibility of a population-based screening for FH in children in Bavaria, intending to set the stage for a nationwide FH screening infrastructure. Furthermore, we aim to validate genetic variants of unclear significance, detect novel causative mutations and contribute to polygenic risk indices (DRKS00022140; August 2020). [ABSTRACT FROM AUTHOR]

Introduction: MicroRNAs play an important role in many human malignancies; so far, their expression remains to be studied in upper urinary tract urothelial cancer (UUTUC). Materials and Methods: The expression of eleven microRNAs (miR-10a, miR-21, miR-96, miR-135, miR-141, miR-182, miR-200b, miR-205, miR-429, miR-520b, miR-1244) formerly shown to be upregulated in urothelial bladder cancer were studied in corresponding normal and cancerous tissue samples of patients undergoing nephroureterectomy for UUTUC. Upregulated microRNAs were then measured in serum samples of patients with UUTUC and patients with non-malignant urological diseases to evaluate their potential as non-invasive biomarkers for UUTUC. Results: MicroRNA expression allowed differentiation of normal and cancerous tissue: miR-21, miR-96, miR-135, miR-141, miR-182, miR-205, miR-429 and miR-520b were significantly overexpressed. Furthermore, miR-205 was upregulated in poorly differentiated UUTUC. The analysis of circulating RNA in serum demonstrated an increase of miR-141 in patients with UUTUC; receiver operator characteristic analysis demonstrated an area under the curve of 0.726 for miR-141 as a diagnostic biomarker. Furthermore, we observed lower levels of miR-10a and miR-135 in UUTUC patients. Conclusions: MicroRNA expression is altered in UUTUC. The analysis of circulating miR-141 may be useful to identify patients with UUTUC. [ABSTRACT FROM AUTHOR]

Recent studies have demonstrated the differential expression of miR-141 and miR-375 in circulation of patients with advanced/metastatic prostate cancer (PCa). The aim of this study was to investigate the regulation of miR-141 and miR-375 by androgens and their release into the incubation medium in relation to prostate specific antigen (PSA) mRNA and prostate cancer antigen 3 (PCA3). Plasma levels of these molecules were measured in a small cohort of patients with localized PCa. As an in vitro cell model we used androgen sensitive LNCaP cells exposed to an androgen ablation of 48h, and then treated with dihydrotestosterone (DHT) for 24h. Expression of the four RNA molecules was measured by quantitative polymerase chain reaction (qPCR). miR 141 and miR 375 were induced in a dose-dependent manner where the median stimulation reached only 1.5 fold at maximum. The effect of DHT on PSA mRNA (up to 30-fold) and PCA3 (up to 195-fold) was much more evident. With regard to the release into the incubation medium, similar results were obtained with the exception of PCA3. At the highest DHT dose (100nM), median miR-141 and miR-375 release was increased 1.7- and 1.4-fold (P=0.07), respectively. DHT treatment led to a significant release of PSA mRNA (up to 12 fold) into the medium while PCA3 could not be amplified from the incubation medium. In plasma only PCA3 differed significantly between localized PCa patients and healthy subjects. In conclusion, our study provides evidence that miR-141 and miR-375 are increasingly released into incubation medium from androgen-stimulated cells. However, the extent of their induction was weaker than PSA mRNA or PCA3, suggesting differential regulation by androgens. [ABSTRACT FROM AUTHOR]

Although prostate-specific antigen-based prostate cancer screening had a positive impact in reducing prostate cancer mortality, it also led to overdiagnosis, overtreatment and a significant number of unnecessary biopsies. In the post-prostate-specific antigen era, new biomarkers have emerged that can complement the information given by prostate-specific antigen, towards a better cancer diagnostic specificity, and also allowing a better estimate of the aggressiveness of the disease and its clinical outcome. That means those markers have the potential to assist the clinician in the decision-making processes, such as whether or not to perform a biopsy, and to make the best treatment choice among the new therapeutic options available, including active surveillance in lower risk disease. In this article, we will review several of those more recent diagnostic markers (4Kscore®, [-2]proPSA and Prostate Health Index, SelectMDx®, ConfirmMDx®, Progensa® Prostate Cancer Antigen 3, Mi-Prostate Score, ExoDx™ Prostate Test, the Stockholm3 test and ERSPC risk calculators) and prognostic markers (OncotypeDX® Genomic Prostate Score, Prolaris®, Decipher® and ProMark®). We will also address some new liquid biopsy approaches – circulating tumour cells and cell-free DNA – with a potential role in metastatic castration-resistant prostate cancer and will briefly give some future perspectives, mostly outlooking epigenetic markers. [ABSTRACT FROM AUTHOR]

Background: The prognostic value of in‐hospital haemoglobin drop in patients with acute coronary syndrome (ACS) undergoing invasive therapy remains insufficiently investigated. Materials and Methods: This observational study included 3838 patients with ACS with admission and in‐hospital nadir haemoglobin values available. Haemoglobin drop was defined as a positive difference between admission and nadir haemoglobin values. The primary endpoint was one‐year all‐cause mortality. Results: In‐hospital haemoglobin drop occurred in 3142 patients (82%). Patients were categorized into 4 groups: no haemoglobin drop (n = 696 patients), <3 g/dl haemoglobin drop (n = 2703 patients), 3 to <5 g/dl haemoglobin drop (n = 344 patients) and ≥5 g/dl haemoglobin drop (n = 95 patients). The primary endpoint occurred in 156 patients: 17 patients (2.5%) in the group with no haemoglobin drop, 81 patients (3.0%) in the group with <3g/dl haemoglobin drop, 37 patients (10.9%) in the group with 3 to <5 g/dl haemoglobin drop and 21 patients (22.2%) in the group with ≥5 g/dl haemoglobin (adjusted hazard ratio [HR] = 1.30, 95% confidence interval 1.17 to 1.45; p <.001 for one g/dl haemoglobin drop). The association of haemoglobin drop with one‐year mortality remained significant after exclusion of patients with in‐hospital overt bleeding (adjusted HR = 1.27 [1.11–1.46]; p <.001 for one g/dl haemoglobin drop). The lowest haemoglobin drop associated with mortality was 1.23 g/dl in all patients (HR = 1.03 [1.02–1.04]) and 1.13 g/dl in patients without overt bleeding (HR = 1.03 [1.01–1.04]). Conclusions: In patients with ACS, in‐hospital haemoglobin drop was associated with higher risk of one‐year mortality even in the absence of overt bleeding. [ABSTRACT FROM AUTHOR]

Invasive mechanical ventilation and oxygen toxicity are postnatal contributors to chronic lung disease of prematurity, also known as bronchopulmonary dysplasia (BPD). Cyfra 21-1 is a soluble fragment of cytokeratin 19, which belongs to the cytoskeleton stabilizing epithelial intermediate filaments. As a biomarker of structural integrity, Cyfra 21-1 might be associated with airway injury and lung hypoplasia in neonates. Serum Cyfra 21-1 concentrations for 80 preterm and 80 healthy term newborns were measured within 48 h after birth. Preterm infants with the combined endpoint BPD/mortality had significantly higher Cyfra 21-1 levels compared with those without fulfilling BPD/mortality criteria (P = 0.01). Also, severe RDS (>grade III) was associated with higher Cyfra levels (P = 0.01). Total duration of oxygen therapy was more than five times longer in neonates with high Cyfra 21-1 levels (P = 0.01). Infants with higher Cyfra 21-1 values were more likely to receive mechanical ventilation (50% vs. 17.5%). However, the duration of mechanical ventilation was similar between groups. The median Cyfra value was 1.93 ng/mL (IQR: 1.68-2.53 ng/mL) in healthy term neonates and 8.5 ng/mL (IQR: 3.6-16.0 ng/mL) in preterm infants. Using ROC analysis, we calculated a Cyfra cutoff > 8.5 ng/mL to predict BPD/death with an AUC of 0.795 (P = 0.004), a sensitivity of 88.9%, and a specificity of 55%. Mortality was predicted with a cutoff > 17.4 ng/mL (AUC: 0.94; P = 0.001), a sensitivity of 100%, and a specificity of 84%. These findings suggest that Cyfra 21-1 concentration might be useful to predict poor outcome in premature infants. [ABSTRACT FROM AUTHOR]

A letter to the editor is shown in response to the article "Serum Tumor Marker Use in Patients with Advanced Solid Tumors."

In pancreatic cancer (PC) accurate determination of treatment response by imaging often remains difficult. Various efforts have been undertaken to investigate new factors which may serve as more appropriate surrogate parameters of treatment efficacy. This review focuses on the role of carbohydrate antigen 19-9 (CA 19-9) as a prognostic tumor marker in PC and summarizes its contribution to monitoring treatment efficacy. We undertook a Medline/PubMed literature search to identify relevant trials that had analyzed the prognostic impact of CA 19-9 in patients treated with surgery, chemoradiotherapy and chemotherapy for PC. Additionally, relevant abstract publications from scientific meetings were included. In advanced PC, pretreatment CA 19-9 levels have a prognostic impact regarding overall survival. Also a CA 19-9 decline under chemotherapy can provide prognostic information for median survival. A 20% reduction of CA 19-9 baseline levels within the first 8 weeks of chemotherapy appears to be sufficient to define a prognostic relevant subgroup of patients (‘CA 19-9 responder’). It still remains to be defined whether the CA 19-9 response is a more reliable method for evaluating treatment efficacy compared to conventional imaging. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

To evaluate the diagnostic performance of the prostate health index (PHI) and of the percentage of [-2]proPSA (%[-2]proPSA) calculated with total and free PSA from non-Beckman Coulter manufacturers (Roche and Abbott), and compare it with the fully Beckman Coulter [-2]proPSA derivatives. In this study, 237 men (PSA: 2–10 μg/L) scheduled for prostate biopsy were enrolled. %[-2]proPSA and PHI were calculated with total and free PSA from three manufacturers. Beckman Coulter PSA and [-2]proPSA were performed on the Access 2 analyzer (Hybritech calibration). Roche PSA was performed on the cobas e411 and the Abbott PSA on the Architect i2000sr. Statistical analysis was performed, considering prostate cancer (PCa) as the outcome. Univariate analysis showed that all indices were predictors of cancer, irrespective of the manufacturer (p<0.001). The AUC was similar for all manufacturers, both for %[-2]proPSA (Beckman Coulter: 0.756; Roche: 0.770; Abbott: 0.756) and PHI (Beckman Coulter: 0.776; Roche: 0.785; Abbott: 0.778). When considering the cutoffs that allowed 90% sensitivity, [-2]proPSA derivatives calculated with Roche and Abbott PSA had similar specificities and predictive values when compared to Beckman Coulter. The percentage of missed cancers (8–9%) was the same between manufacturers. The percentage of spared biopsies was significantly higher with Roche's PHI (21.0%) and Abbott's PHI (20.6%) than with Beckman Coulter's PHI (17.2%). In the PSA range between 2 and 10 μg/L, [-2]proPSA derivatives maintain their diagnostic performance in PCa detection when calculated with PSA from Roche and Abbott. This can lead to a broader implementation of these indices in clinical laboratories worldwide. [ABSTRACT FROM AUTHOR]

• PHI density has a better diagnostic performance than PHI in overall prostate cancer. • For clinically significant cancer PHI and its density perform almost equally. • PSA density performs better than PHI density in detecting significant cancer. Overdiagnosis of prostate cancer (PCa) should be minimized. We wanted to evaluate the diagnostic performance of the prostate health index density (PHID) and compare it with that of the prostate health index (PHI) alone and of the prostate-specific antigen density (PSAD). 232 men scheduled for a prostate biopsy (prostate-specific antigen level: 2–10 µg/L), were enrolled. PHI, PHID and PSAD were evaluated considering PCa and clinically significant PCa (csPCa) as the outcomes. For PCa, the area under the curve (AUC) was higher for PHID (0.823) than for PHI (0.779) and PSAD (0.776). For csPCa, the AUC was also higher for PHID (0.851) but closer to that of PSAD (0.819) and PHI (0.813). For equal sensitivities (90%) for PCa, PHID and PSAD offered the highest specificities (37%), missing the same number of cancers (n = 11). Considering csPCa, PHI and PHID had similar specificities. PSAD reached the highest specificity (50.0%), sparing 32.8% of biopsies, while missing 9 cases of csPCa. PHID has a better diagnostic performance than PHI for overall PCa detection, but very close to the PSAD performance. Considering csPCa, PHI and PHID perform almost equally, but PSAD has a better diagnostic performance. [ABSTRACT FROM AUTHOR]

Acute type A aortic dissection (ATAAD) constitutes a life-threatening aortic pathology with significant morbidity and mortality. Without surgical intervention the usual mortality rate averages between 1 and 2% per hour. Thus, an early diagnosis of ATAAD is of pivotal importance to direct the affected patients to the appropriate treatment. Preceding tests to find an appropriate biomarker showed among others an increased aggrecan (ACAN) mRNA expression in aortic tissue of ATAAD patients. As a consequence, we investigated whether ACAN is a potential biomarker for diagnosing ATAAD. Mean ACAN protein concentration showed a significantly higher plasma concentration in ATAAD patients (38.59 ng/mL, n = 33) compared to plasma of patients with thoracic aortic aneurysms (4.45 ng/mL, n = 13), patients with myocardial infarction (11.77 ng/mL, n = 18) and healthy volunteers (8.05 ng/mL, n = 12). Cardiac enzymes like creatine kinase MB and cardiac troponin T showed no correlation with ACAN levels in ATAAD patients. Receiver-operator characteristics (ROC) curve analysis for ATAAD patients versus control subjects an optimum discrimination limit of ACAN plasma levels at 14.3 ng/mL with a corresponding sensitivity of 97% and specificity of 81%. According to our findings ACAN is a reliable potential biomarker in plasma samples to detect ATAAD with high sensitivity and specificity. [ABSTRACT FROM AUTHOR]

Recent advances in basic research have unveiled several strategies for improving the sensitivity and specificity of cell-free DNA (cfDNA) based assays, which is a prerequisite for broadening its clinical use. Included among these strategies is leveraging knowledge of both the biogenesis and physico-chemical properties of cfDNA towards the identification of better disease-defining features and optimization of methods. While good progress has been made on this front, much of cfDNA biology remains uncharted. Here, we correlated serial measurements of cfDNA size, concentration and nucleosome histone modifications with various cellular parameters, including cell growth rate, viability, apoptosis, necrosis, and cell cycle phase in three different cell lines. Collectively, the picture emerged that temporal changes in cfDNA levels are rather irregular and not the result of constitutive release from live cells. Instead, changes in cfDNA levels correlated with intermittent cell death events, wherein apoptosis contributed more to cfDNA release in non-cancer cells and necrosis more in cancer cells. Interestingly, the presence of a ~ 3 kbp cfDNA population, which is often deemed to originate from accidental cell lysis or active release, was found to originate from necrosis. High-resolution analysis of this cfDNA population revealed an underlying DNA laddering pattern consisting of several oligo-nucleosomes, identical to those generated by apoptosis. This suggests that necrosis may contribute significantly to the pool of mono-nucleosomal cfDNA fragments that are generally interrogated for cancer mutational profiling. Furthermore, since active steps are often taken to exclude longer oligo-nucleosomes from clinical biospecimens and subsequent assays this raises the question of whether important pathological information is lost. [ABSTRACT FROM AUTHOR]

Cell-free DNA (cfDNA) has become widely recognized as a promising candidate biomarker for minimally invasive characterization of various genomic disorders and other clinical scenarios. However, among the obstacles that currently challenge the general progression of the research field, there remains an unmet need for unambiguous universal cfDNA nomenclature. To address this shortcoming, we classify in this report the different types of cfDNA molecules that occur in the human body based on its origin, genetic traits, and locality. We proceed by assigning existing terms to each of these cfDNA subtypes, while proposing new terms and abbreviations where clarity is lacking and more precise stratification would be beneficial. We then suggest the proper usage of these terms within different contexts and scenarios, focusing mainly on the nomenclature as it relates to the domains of oncology, prenatal testing, and post-transplant surgery surveillance. We hope that these recommendations will serve as useful considerations towards the establishment of universal cfDNA nomenclature in the future. In addition, it is conceivable that many of these recommendations can be transposed to cell-free RNA nomenclature by simply exchanging "DNA" with "RNA" in each acronym/abbreviation. Similarly, when describing DNA and RNA collectively, the suffix can be replaced with "NAs" to indicate nucleic acids. [ABSTRACT FROM AUTHOR]

• Available cancer biomarkers are associated with low diagnostic performance. • Satellite 2 DNA amount is higher in the blood circulation of patients with cancer. • Satellite 2 DNA may be a universal biomarker for the detection of cancer. Currently, cancer biomarkers are associated with low diagnostic performance, and the notion of using cell-free DNA (cfDNA) as a surrogate cancer biomarker is a subject of ongoing research efforts. Pericentromeric satellite repeats were shown to expand in tumor cells. Here, we hypothesized that the increased release of satellite DNA into the circulation might be a basis for developing a biomarker for the detection of cancer. The study included patients with different cancer types, and controls without cancer. Human satellite 2 repeat (HSATII) from chromosomes 1, 10, or 16 was amplified using extracted DNA from plasma or direct application of diluted plasma in PCR. We first showed that HSATII DNA levels were higher in patients with cancer than in controls relative to LINE1 element, with chr10-HSATII being the most relevant. Absolute quantification in digital PCR showed much higher levels of chr10-HSATII in patients with breast cancer compared with healthy individuals. Subsequently, employing diluted plasma also revealed that HSATII DNA was present in increased levels in patients with cancer including breast, gastric, lung or bile cancers, sarcoma or Hodgkin's lymphoma than in controls with an AUC of 94% in the ROC curve. This proof-of-concept study reveals the high potential of HSATII DNA as a surrogate cancer biomarker. [ABSTRACT FROM AUTHOR]

Keywords: evidence based-medicine; reference change value; tumor biomarker EN evidence based-medicine reference change value tumor biomarker 553 555 3 03/11/22 20220301 NES 220301 To the Editor, A word of caution on using biomarker reference change values (RCV) to guide medical decisions: In a recent study from the EFLM working group on biological variation, the biological variation of tumor markers has been investigated and estimates of the within and between subject biological variations were obtained [[1]]. Also, cancer treatment itself has a significant effect on the tumor and therefore tumor biomarker dynamics. [Extracted from the article]

Through various pathways of cell death, degradation, and regulated extrusion, partial or complete genomes of various origins (e.g., host cells, fetal cells, and infiltrating viruses and microbes) are continuously shed into human body fluids in the form of segmented cell-free DNA (cfDNA) molecules. While the genetic complexity of total cfDNA is vast, the development of progressively efficient extraction, high-throughput sequencing, characterization via bioinformatics procedures, and detection have resulted in increasingly accurate partitioning and profiling of cfDNA subtypes. Not surprisingly, cfDNA analysis is emerging as a powerful clinical tool in many branches of medicine. In addition, the low invasiveness of longitudinal cfDNA sampling provides unprecedented access to study temporal genomic changes in a variety of contexts. However, the genetic diversity of cfDNA is also a great source of ambiguity and poses significant experimental and analytical challenges. For example, the cfDNA population in the bloodstream is heterogeneous and also fluctuates dynamically, differs between individuals, and exhibits numerous overlapping features despite often originating from different sources and processes. Therefore, a deeper understanding of the determining variables that impact the properties of cfDNA is crucial, however, thus far, is largely lacking. In this work we review recent and historical research on active vs. passive release mechanisms and estimate the significance and extent of their contribution to the composition of cfDNA. [ABSTRACT FROM AUTHOR]

Background and Purpose: Irinotecan, used in colorectal cancer therapy, is metabolized by glucuronidation involving different UDP-glucuronosyltransferase (UGT)1A isoforms leading to facilitated elimination from the body. Individuals homozygous for the genetic variants UGT1A1*28 (Gilbert syndrome) and UGT1A7*3 are more susceptible to irinotecan side effects, severe diarrhoea and leukopenia. The aim of this study was to investigate the protective effects and active constituents of coffee during irinotecan therapy using humanized transgenic (htg)UGT1A-WT and htgUGT1A-SNP (carry UGT1A1*28 and UGT1A7*3 polymorphisms) mice.Experimental Approach: HtgUGT1A mice were pretreated with coffee or caffeic acid (CA) + caffeic acid phenylethyl ester (CAPE) and injected with irinotecan. The effects of coffee and CA + CAPE were investigated using reporter gene assays, immunoblot, TaqMan-PCR, siRNA analyses and blood counts.Key Results: Only the combination of the two coffee ingredients, CA and CAPE, mediates protective effects of coffee in a model of irinotecan toxicity by activation of UGT1A genes. Coffee and CA + CAPE significantly increased UGT1A expression and activity along with SN-38 glucuronide excretion in irinotecan-injected htgUGT1A mice, resulting in significant improvement of leukopenia, intestinal oxidative stress and inflammation.Conclusion and Implications: In this study, we identify the compounds responsible for mediating the previously reported coffee-induced activation of UGT1A gene expression. CA and CAPE represent key factors for the protective properties of coffee which are capable of reducing irinotecan toxicity, exerting antioxidant and protective effects. Provided that CA + CAPE do not affect irinotecan efficacy, they might represent a novel strategy for the treatment of irinotecan toxicity. [ABSTRACT FROM AUTHOR]

Background: NT-proBNP is one of the most important biomarkers for the diagnosis and risk assessment of heart failure in adults. Age- and gender-independent reference intervals (RIs) have been reported. In contrast, RIs in children are strongly age-dependent, do not exist for all ages and reveal a right-skewed distribution. Accordingly, no common Z-score can be formed and a cross-age interpretive method, so far, is missing. Methods: Within the paper on hand, new evaluation techniques are applied to already published NT-proBNP study results and additionally to newly gained data. Upper limits (ULs), lower limits (LLs) and 50th percentiles are tested for power-like behavior as a function of age using linear regression analysis. Functions for continuous RIs are derived and reference limits are calculated on a per day basis. A corresponding Zlog formula is deduced and its usefulness is stated in two clinical examples. Results: The power-like behavior of NT-proBNP concentration from birth to 18 years is demonstrated. With age in days t and measured NT-proBNP value x in pg/mL, an age-specific Zlog value may directly be calculated using the equation: Zlog NT-proBNP = log x + 0.512 ⋅ log t − 3.417 1.489 + 0.014 ⋅ log t ⋅ 3.92 ${\rm{Zlo}}{{\rm{g}}_{{\rm{NT - proBNP}}}} = {{\log \;x + 0.512 \cdot \log \;t - 3.417} \over {1.489 + 0.014 \cdot \log \;t}} \cdot 3.92$ Conclusions: Using formulas for UL and LL, continuous RIs from 0 to 18 years may be obtained. Continuity corresponds to physiological changes in the body much better than discrete RIs. With the advent of an NT-proBNP-specific Zlog value, a cross-age Z-score equivalent is providing an easy interpretation aid in everyday pediatric practice. This new approach allows to identify clinical worsening much better, sooner and more clearly than previous absolute values. [ABSTRACT FROM AUTHOR]

Introduction: Alterations in cell-free DNA concentration (cfDNA) over time have been studied in diseased or injured patients or analyzed in athletes during exhaustive exercise. However, no fluctuations have been examined over a short time course in healthy humans at rest so far, wherefore the aim of this study was to examine individual variations at different time points within 75 min. Methods: Serial blood drawing was performed in 14 healthy female volunteers at rest within 75 min. Plasma DNA was quantified by real-time qPCR, and absolute levels were analyzed together with relative variations. cfDNA alterations were moreover analyzed in consideration of potential volunteer-related impact factors (e.g., pulse) and were compared to alterations of plasma CK and AST. Results: Absolute cfDNA concentration ranged from 0.6 to 3.4 ng/ml. Regarding alterations over time, positive and negative variations were identified, whereby the interdecile range of fold changes was from 0.5 to 1.4. The maximum fold change was determined at 10 min. No relations were found between cfDNA levels and the analyzed individual factors. Conclusion: We evidenced the variability of cfDNA in healthy humans at rest within a short time course. The determined variations should serve in future studies to distinguish small cfDNA increases after minor trauma from natural fluctuations. Without such reference of intra-individual variation at rest, it would not be feasible to distinguish an injury from a fluctuation with certainty. Thus, a basis was established for the application of cfDNA as biomarker for the detection of mild injuries in forensic biomechanics. [ABSTRACT FROM AUTHOR]

Pulmonary hypertension (PH) and lung hypoplasia are major contributors to morbidity and mortality in newborns with congenital diaphragmatic hernia (CDH). The soluble receptor for advanced glycation end products (sRAGE) is a marker of endothelial function and might be associated with disease severity in CDH newborns. In a cohort of 30 CDH newborns and 20 healthy control newborns, sRAGE concentration was measured at birth and at 6 h, 12 h, 24 h, 48 h, and 7–10 days. In healthy newborns, sRAGE was significantly higher at birth and at 48 h compared with CDH newborns (both P < 0.001). Among CDH newborns, sRAGE was significantly lower at birth (P = 0.033) and at 7–10 days (P = 0.035) in patients receiving extracorporeal membrane oxygenation (ECMO) compared with patients not receiving ECMO. In contrast, CDH newborns receiving ECMO had significantly higher values at 6 h (P = 0.001), 12 h (P = 0.004), and 48 h (0.032). Additionally, sRAGE correlated significantly with PH severity, intensity and duration of mechanical ventilation, and prenatally assessed markers of CDH severity (lung size, liver herniation). The probability to receive ECMO therapy was five times higher in CDH newborns with sRAGE concentrations below the calculated cutoff of 650 pg/ml at birth (P = 0.002) and nine times higher in CDH newborns with sRAGE concentrations above the cutoff of 3,500 pg/ml at6h(P = 0.001). These findings suggest a potential involvement of sRAGE in the pathophysiology of CDH and may act as a therapeutic target in future treatment approaches. [ABSTRACT FROM AUTHOR]